Dr. Cheng Zhu (Georgia Institute of Technology)

Committee:

Dr. Susan Thomas (Georgia Institute of Technology)

Dr. Michelle Krogsgaard (New York University)

Dr. Mandy Ford (Emory University)

Dr. Gabe Kwong (Georgia Institute of Technology)

Regulation of T-cell antigen recognition by melanoma tumor microenvironment and TCR-CD3 ectodomain interaction

Despite the critical role of CD8+ T cells in tumor clearance, their functions are impaired by immunosuppressive cells/cytokines, through inhibitory receptors, and metabolic restrictions in the tumor microenvironment (TME). Targeting these suppressive pathways were shown to promote tumor clearance, yet unknown mechanisms may still exist curtailing the T cell responses. The T cell activation and anti-tumor response are initiated by the T cells receptor (TCR) recognizing antigen peptide presented by major histocompatibility complex (pMHC) molecules on antigen presenting cells (APC). Recent studies have demonstrated that comparing to in solution (or three-dimensional, 3D) kinetic measurements that uses purified TCR molecules, analysis of pMHC interacting with TCRs expressed on native T cells (or two-dimensional, 2D) provides a better prediction of T cell function and is able to capture perturbations of antigen recognition by T cell intrinsic and extrinsic mechanisms. In this study, we examined whether T cell antigen recognition is altered by the TME, and thus contributes to the T cell dysfunction. By testing the OT-I T cells from the murine B16F10 melanoma TME with their cognate antigen pMHC OVA:H2Kb, we showed that the TCR-pMHC 2D affinity is reduced in TME. The presence of tumor modulated TCR mechanosensing of antigen pMHC, converting a typical TCR-pMHC catch bond into slip bonds. The T cells from TME gave a reduced spreading on pMHC coated surface, with a decreased TCR-pMHC tension signal generated by spontaneous T cell pulling on pMHC at force over 4.7pN. The TME altered dynamic response of T cell CD3ζ phosphorylation, and reduced level of calcium flux following in vitro stimulations. Using T cell in vitro activation, in vivo proliferation and ex vivo cytokine production as readouts, we showed that removing the TME restores T cell function that was impaired by this antigen inexperienced mechanism. Further analysis showed that nitration of TCR, which can be caused by presence of MDSCs in TME and induces T cell tolerance induced dysfunction, reduced TCR-pMHC 2D affinity. Presence of immunosuppressive Treg and cytokine TGF-β in TME is known to impair CD8+ T cell activation and function. We showed that in vivo TGF-β inhibition and CD4 depletion in tumor bearing animal partially restored the TME altered TCR-pMHC interaction. To summarize, we found that the impaired TCR-pMHC mechanosensing correlated with a reduced T cell function in TME, while this tumor antigen inexperienced suppression was functionally reversible. We also identified several immunosuppressive factors as the potential mechanisms of TME impairment on T cell antigen recognition.

TCR α chain and β chain bind noncovalently to dimeric subunits CD3δε, CD3γε, and CD3ζζ to form TCR complex. Upon TCRαβ engaging the antigen pMHC, this binding signal is transmitted through TCR-CD3 interaction, phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3 cytoplamic tails, which triggers the T cell activation. The interactions among TCR, CD3δε and CD3γε ectodomains are weak in 2D affinities, with short-moderate duration of averaged lifetimes, however disrupting these interactions affects TCR complex stability, signaling and significantly reduces T cell function. In this study, we examed how this weak TCR-CD3 extracellular interaction severely impacts T cell function and whether this impact is through regulating T cell antigen recongition. We found that purified TCR proteins bind to a mixture of CD3δε and CD3γε ectodomain proteins at an increased likelihood and increased average lifetime, comparing to TCR-CD3δε or TCR-CD3γε interaction. This proved the existence of cooperativity among TCR-CD3 extracellular domain interactions. The antibody/Fab targeting TCR and CD3 ectodomains can block TCR-CD3 extracellular interaction and reduce T cell functional response. We showed that the TCR-CD3 interaction blocking Fab treatments decreased TCR-pMHC 2D affinity and altered TCR-pMHC interaction force response profile. Together, these results indicate that TCR-CD3 extracellular interactions is enhanced by the cooperativity among the ectodomain interactions, and regulates T cell function through altering TCR mechanosensing.

In this study, we identified impaired T cell antigen recognition as one mechanism of T cell dysfunction in TME. We also identified the cooperativity enhanced TCR-CD3 extracellular interaction as a regulating factor of T cell function by affecting T cell antigen recognition. The results greatly extend our understanding on how the T cell antigen recognition is regulated in physiological and pathological conditions, providing the molecular basis for developing pharmacological approaches to restore/promote or suppress T cell response by regulating T cell antigen recognition.